The objective of this project is to develop a human HIV-1 genetic (DNA) vaccine for global application that will include the following elements of an effective vaccine: (1) HIV-1 sequences that bind to supertype human lymphocyte antigens (HLA) that represent the majority of global human populations. (2) HIV-1 sequences that represent the global population of HIV-1 genotypes and all the major clades. (3) A vaccine that stimulates all arms of the immune system and elicits immune memory to HIV challenge. The research design is based on DNA vaccine chimeras with selected HIV-1 T cell epitope sequences inserted into the luminal domain of the lysosome-associated membrane protein (LAMP) and targeted to organelles of antigen presenting cells (ARC) that contain major histocompatability class II (MHC II) proteins for presentation of antigen epitopes to CD4+ T-cells and activation of immune memory. Targeting of endogenous antigens to the MHC II compartment is known to stimulate both T- and B-cell responses and immune memory. Epitope selection is conducted by computer modeling of all HIV-1 genome sequences with algorithms that select protein sequences that are conserved in clades A to D and contain multiple, mostly overlapping, nonameric peptide sequences (hotspots) that bind to HLA supertype alleles. These supertypes represent groups of HLA alleles that have subtle differences in their binding grooves and are present in a large proportion of the human population. Validaton of T-cell activation by the selected sequences is conducted by immunizing HLA transgenic mice with DNA constructs encoding the MHC II- targeted sequences and analyzing epitope-specific T-cell responses with overlapping peptides spanning the epitope hotspot sequences. Further correlation of the selected HIV-1 sequences to human immune responses will be in collaboration with other investigators to carry out ELISpot analysis of the ex vivo T-cell responses to the selected peptide sequences of leucocytes of patients infected by HIV-1 of all major clades. Additionally, the biological role of the selected epitope sequences will be analyzed with a mouse model yellow fever virus (YFV) challenge assay system. The protective effect of immunization with the LAMP/HIV epitope chimera will be assayed by challenging the immunized animal with YFV containing the same HIV sequence. Relevance: This project proposes the application of novel technologies to the development of an effective HIV-1 vaccine applicable to global populations and all major HIV-1 clades, and capable of eliciting immune memory.